Transcription, splicing and editing of plastid RNAs in the...

Plant Molecular Biology 29: 721-733, 1995. © 1995 Kluwer Academic Publishers. Printed in Belgium. 721

Transcription, splicing and editing of plastid RNAs in the nonphotosynthetic plant Epifagus virginiana

Stephanie C. Ems 1, Clifford W. Morden 2, Colleen K. Dixon 3, Kenneth H. Wolfe4, Claude W. dePamphilis 5 and Jeffrey D. Palmer* Department of Biology, Indiana University, Bloomington, IN 47405, USA (* author for correspondence); present addresses: 1Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202, USA; 2Department of Botany/H.E.B.P., University of Hawaii at Manoa, Honolulu, HI 96822, USA; 3Eli Lilly and Company, Inc., Lilly Corporate Center, Indianapolis, IN 46285, USA. 4Department of Genetics, University of Dublin, Trinity College, Dublin 2, Ireland," 5 Department of Biology, Vanderbilt University, Nashville, TN 37235, USA

Received 17 May 1995; accepted in revised form 11 August 1995

Key words: Epifagus, maturase, nonphotosynthetic plant, RNA editing, splicing, transcription

Abstract

Expression of the vestigial plastid genome of the nonphotosynthetic, parasitic flowering plant Epifagus virginiana was examined by northern analysis and by characterization of cDNAs. Probes for each of 12 plastid genes tested hybridized to all lanes of northern blots containing total RNA prepared from stems and fruits of Epifagus and from leaves of tobacco. Certain transcript patterns in Epifagus plastids are highly complex and similar to those of tobacco operons. In contrast, genes such as rps2, which have become orphaned in Epifagus as a result of evolutionary loss of formerly cotranscribed genes, show simpler transcript patterns in Epifagus than in tobacco. Sizing and sequencing of cDNAs generated by reverse transcriptase-PCR for three genes, rpsl2, rpl2, and clpP, show that their transcripts are properly cis- and/or trans-spliced at the same five group II intron insertion sites used in photosynthetic plants. A single, conventional C ~ U edit in rpsl2 was found among the total of 1401 nucleotides of cDNA sequence that was determined for the three genes. An octanucleotide sequence identical to a putative guide RNA of plant organelles and perfectly complementary to the rpsl2 edit site itself was identified just 200 bp upstream of the edit site. These data, together with previous results from the complete sequencing of the Epifagus plastid genome, provide compelling evidence that this degenerate genome is nonetheless expressed and functional. Analysis of the putative maturase MatK, encoded by the group II intron of trnK in photosynthetic land plants but by a freestanding gene in Epifagus, leads us to hy- pothesize that it acts 'in trans' to assist the splicing of group II introns other than the one in which it is normally encoded.

Introduction

Complete sequencing of the 70 kb plastid genome of the nonphotosynthetic, parasitic flowering

plant Epifagus virginiana (beechdrops; Oroban- chaceae) has shown that it contains only 42 intact genes [27, 41-44], compared to 113 plastid genes in tobacco, its closest, fully sequenced photosyn-

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thetic relative [32, 34, 40]. Although at least 38 of the 42 Epifagus plastid genes specify components of the gene expression apparatus, all four normally plastid-encoded RNA polymerase genes are missing or defunct, implying that plastid transcription in Epifagus is entirely dependent on a nuclear-encoded polymerase [27, 41 ]. All but one of these 38 gene expression genes encode components of the plastid translational apparatus. However, many plastid tRNA and ribosomal protein genes have also been lost from the Epifa- gus genome, again implying surprising reliance on nuclear gene products [27, 42]. Functions are known for only two of the four nongenetic genes in Epifagus, clpP (encoding a protease subunit) and accD (encoding a subunit of acetyl-CoA car- boxylase).

Surprisingly, the one nontranslational gene thought to be involved in gene expression, which we previously named 'matK', is a freestanding gene in Epifagus [41]. In contrast, in all examined photosynthetic land plants matK is contained within the group II intron of the tRNA-lysine (UUU) gene (trnK) and is thought to encode a 'maturase', or splicing factor, that assists in the splicing of the intron in which it residues [2, 3, 11, 21, 28, 31, 33, 37, 38]. In particular, Mohr etal. [26] have shown that all matK genes contain a ca. 100 amino acid domain (termed domain 'X') that is present in virtually all group II intron maturases. We [41] and others [8, 22, 26] have sug- gested that, uniquely among group II maturases, the matK product (MatK) assists splicing of multiple, distantly related group II introns.

Molecular evolutionary evidence suggests that the Epifagus plastid genome is expressed and functional [41]. First, deletions are highly non- random; for example, 95 ~o of photosynthetic sequences have been deleted, whereas only 20~o of ribosomal protein sequences and none of the ribosomal RNA sequences are missing. Second, whereas 71 genes are entirely or largely deleted, many of the 42 remaining intact genes are so large (e.g., 2216 and 1738 codons) that their mainte- nance must result from selection for their continued function.

While the evolutionary arguments in favor of

plastid genome function in Epifagus are strong, evidence in the form of detectable gene products is minimal. Thus far, this consists of a single northern blot of Epifagus total RNA, to which a probe for the 16S rRNA gene from tobacco chloroplasts hybridized to transcripts of 1.5 kb (interpreted as plastid 16S rRNA) and 1.8 kb (interpreted as cross-hybridization to mitochondrial 18S rRNA) [4]. A major goal of the present study is to remedy this situation by determining whether or not transcripts can be detected from a significant number of Epifagus plastid genes. A second goal is to examine the pattern of plastid transcripts in Epifagus, and to make direct comparisons to tobacco, in order to investigate the effects of the many gene and promoter deletions [41] on the expression of plastid operons of varying complexity. A final goal is to determine whether group II introns are properly spliced in Epifagus plastids and to relate this to the putative maturase activity of its freestanding matK gene.

Materials and methods

Above-ground stems and fruits of mature plants of Epifagus virginiana were collected from locations in Michigan, Indiana and Tennessee. Leaves of tobacco (Nicotiana tabacum) were harvested from greenhouse-grown plants. Field-collected plants were frozen in liquid nitrogen upon collec- tion and stored at -70 °C prior to nucleic acid isolation. Total DNA was isolated using the modified CTAB method of Doyle and Doyle [5 ]. Total RNA was isolated either as described previously [4] or according to the following procedure. Frozen tissue (5-7 g) is ground in liquid nitrogen using a chilled mortar and pestle, and then placed in a -70 ° C freezer for 5 min to drive offthe liquid nitrogen. The frozen powder is added to a hot (60 °C) phenol emulsion [10 ml phenol plus 12 ml extraction buffer (100 mM Tris pH 8.0, 2 0 m M EDTA, 0 .4~ SDS, 0.5 M NaC1, 0 .1~ 2-mercaptoethanol)] and stirred at 60 °C for 5 min. Ten ml of chloroform/isobutanol (24:1) is added, and stirring is continued for another 5 min. The solution is centrifuged in an RNase-free tube

at 10000 r.p.m, for 10 min at room temperature, and the aqueous phase is transferred to a new centrifuge tube containing 10 ml of chloroform/ isobutanol (24:1). The solution is mixed well and then centrifuged as above. The aqueous phase is adjusted to 2 M NH4 OAc and precipitated with 1 volume of isopropanol at room temperature for 15 min. The precipitate is collected by centrifuga- tion, washed twice with 70~ ethanol, dried, and resuspended in 0.8 ml of DEPC-treated dHzO.

RNA samples were denatured with glyoxyl and dimethyl sulfoxide and electrophoresed in 1.4~o agarose gels (using a Jordan Scientific recirculat- ing gel apparatus) according to the procedure of Sambrook et al. [30]. RNA was transferred from gels to MagnaGraph nylon filters using a Strat- agene Posiblotter. Filters were UV-crosslinked for 23 s using a DNA transfer lamp from Photo- dyne and baked at 80 °C for 1 h. Hybridization probes were made by random-prime labeling [30] with 32p-dATP. Filters were prehybridized for 4-16 h and hybridized overnight in 1 M NaC1, 1~o SDS at 60 °C. Filters were washed in 2 x SSC, 5~o SDS two times for 5 min at room temperature and then three times for 30 min at 60 ° C. Filters were stripped of probe by two 5 min boil- ing washes with 0.1 x SSC, 0.1~o SDS, 0.2 M Tris-HC1.

PCR reactions were carried out using Taq polymerase from Promega, dNTPs from Pharma- cia, and the following pairs of primers: for rpl2 the 5' primer sequence was ATGGCGATACATT- TATACAAAACTTCTAC [primer coordinates, according to the Epifagus coordinate system ([41]; also see GenBank accession number M81884) are 21406-21378] and the 3' primer was AGCCAACGCTTAGATCCGGCTCTA- CC (coordinates 20 111-20 136); for rps12 the 5' primer was AAAAAGACAGCCAA(AT)C(CA) (12 833-12 817) and the 3' primer was ATAAG- GGCTAAAATCAC (31297-31 313); for 5' clpP the 5' primer was ATGCCTATTGGTGTNCC- NAA (14802-14783) and the 3' primer was AAAGATCCCATTG(AT)NGCNGC (13 844- 13 863); for 3' clpP the 5' primer was CTATAT- CTCAGTAT(AT)GA(AG)GA (14 004-13 985) and the 3' primer was CATAAAAACAT-

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C(AG)CT(AG)TCCAT (13055-13075). Reac- tion volumes and conditions varied depending on use of thermocyclers from either Perkin Elmer Cetus or Idaho Technology. Some reverse- transcriptase-PCR (RT-PCR) reactions were performed using the Perkin Elmer Cetus Gene- Amp RNA PCR kit. In general, the reverse transcription step consisted of incubations at 42 °C for 15 min, 99 °C for 5 min, and 5 °C for 25 min, while PCR consisted of 30 cycles of 95 °C for 1 min, 50 °C for 1 min, and 72 °C for 2min. RNase (RNace-It) and DNase I (RNase-free) were from Stratagene. PCR products were analyzed on 2~o NuSieve (FMC)/I~o agarose gels and sequenced directly by standard methodology.

Results

Transcript patterns in Epifagus plastids

Northern blot analyses were carried out for 12 Epifagus plastid genes to determine whether these genes are expressed, to evaluate the complexity of their transcript patterns, and to make comparisons to the situation in tobacco, its closest relative among well-studied photosynthetic plants. All hybridizations were carried out against filters containing total RNA from stems and fruits of Epifagus and from green leaves of tobacco (e.g. Fig. 1). Preliminary experiments [4] showed that, for most genes, these Epifagus organs contain only about one-fiftieth as much plastid RNA as a proportion of total cellular RNA as do tobacco leaves. Hence, to obtain similar signals across lanes, we loaded 5 #g of each Epifagus RNA but only 0.1 #g of tobacco RNA.

Probes for all 12 Epifagus genes tested hybridized to one or more discrete transcripts, suggest- ing that they are all transcribed. These genes in- clude all four rRNA genes (23S, 16S, 5S, 4.5S), four ribosomal protein genes (rpl2, rps2, rps12, rps14), two large ORFs (0RF2216, 0RF1738), accD, and clpP. The 12 genes can be roughly grouped into three classes according to the complexity of their transcript patterns in Epifagus and the similarity of these pattems between Epifagus

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Fig. 1. Northern blot analysis of plastid transcript patterns in Epifagus and tobacco. A 5/~g portion of total RNA from stems (st) and fruits (fr) of Epifagus (E) and 0.1/~g of total leaf RNA from tobacco (T) were electrophoresed in agarose gels, transferred to filters, and hybridized with PCR probes (see Fig. 2 for probe locations) specific for each of the four genes indicated below the hybridization panels. Transcript sizes (in kb) are based on the 0.24-9.5 kb RNA ladder of GIBCO BRL. The clpP and rps12 probes were hybridized successively to one filter, while the rpl2 and rps2 probes were hybridized to a different filter, made from a gel that had been run less far.

and tobacco. The two large ORFs detected one or two large ( > 6 kb) transcripts of similar size in both species (data not shown). In contrast, two ribosomal protein genes (rps2, Fig. 1; rps14, data not shown) gave far simpler transcript patterns in Epifagus than in tobacco. The remaining eight genes all displayed generally similar and highly complex transcript patterns in both species. Ex- amples of these latter two classes of patterns are illustrated in Fig. 1 and described below.

Transcript patterns for clpP and rps 12

Throughout photosynthetic land plants, including tobacco, exon 1 of rps12 is located between clpP and rps20 and is thought or known to be cotranscribed with them (Fig. 2; [14, 15, 39]). Exons 2 and 3 of rps12 are located in the large inverted repeat, far away from exon 1, and are instead cotranscribed with rps7 (Fig. 2). The mature mRNAs from each of these four genes are created by a complex series of events, including indepen- dent transcription of two small operons, transcript clipping between coding regions, three cis-

splicing events (of intron 2 of rpsl2 and of both introns of clpP), and the trans-splicing of intron 1 of rps12 [10, 15-17, 45]. All four of these genes are retained as intact, conserved open reading frames in Epifagus ptDNA, and their organization is also conserved (Fig. 2; [41, 42]). Probes for two of the four genes (clpP and rps12) gave highly complex patterns of transcripts for both Epifagus and tobacco (Fig. 1). The clpP patterns are more similar between the two species, both qualitatively and quantitatively, than are the rps12 patterns. The six most abundant clpP transcripts are of the same or similar sizes in the two species. The smallest of these, the putative monocistronic, fully spliced clpP m R N A of 800-900 nucleotides (ClpP is 197 amino acids), is, by at least a factor of two, the most abundant transcript in both species. Relatively few of the many rps12 transcripts are of the same size in the two species, and even for these there are significant quantitative differences. Thus, in tobacco there are two predomi- nant rps12 transcripts, of 1.6 kb (the putative 'mature dicistronic rps12/rps7 mRNA'; [ 10]) and of 2.5 kb, whereas in Epifagus these are just two of a multitude of bands of roughly equal abun-

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tobacco

Epifagus

tobacco

Epifagus

dpP 5'rpsl2 rpl20 3'rpsl2 rps7 mr--m m m

I I / - I l

rp.D6 rplT.3 rpl2 rpsl9 rp122 rps3 rpll6 rpU4 rps8 tufa [ rpsll rpoA m n m m ~ m m mm ---m

i i i / / [ ~ i 661 • l i ~ iimmm) .~

Ill

tobacco I

Epifagus

rps2 atpI atpH atpF atpA • • m

Fig. 2. Comparative organization in Epifagus and tobacco of three sets of genes whose expression is examined in Figs. 1 and 3. Filled boxes represent exons, open boxes show introns, and hatched boxes indicate pseudogenes. Angled lines between the Epifagus and tobacco maps connect homologs. Arrows indicate direction of transcription of genes thought to be contranscribed. The sizes of the four cis-spliced Epifagus introns whose splicing is examined in Fig. 3 are indicated in bp within the intron boxes. The two 'halves' of the trans-spliced intron in Epifagus rps12 are indicated with an 'X'. The horizontal lines below the Epifagus maps indicate regions that were PCR-amplified to analyze plastid splicing (Fig. 3) and/or generate probes for northern hybridizations (Fig. 1). The vertical slashes at the ends of these lines indicate the positions of the PCR primers (see Materials and methods for primer coordinates and sequences). The numbers below these lines indicate the expected sizes of PCR products using either genomic DNA as template (before the slashes) or cDNA as template (after the slashes). For rps2, only genomic PCR was done. The four underlined PCR product sizes indicate those products used as probes in Fig. 1. The dashed line and parentheses below rps12 denote the fact that, since intron 1 of rps12 is trans-spliced, its flanking exons, as located in the same orientation, are 51,565 bp apart in the genome. Epifagus mapping data are from [41]; tobacco mapping and expression data are from [ 10, 17, 29, 32, 34, 36, and 45].

dance. As expected for contranscribed genes (Fig. 2), clpP and rpsl2 hybridized to some of the same-sized larger bands, i.e., transcripts of 3.7 and 3.0 kb (Fig. 1).

These comparisons between species are com- plicated by the more subtle differences evident in transcript patterns between Epifagus RNAs made from stems and fruits (Fig. 1). The major clpP transcript in stems and fruits is 0.8 and 0.9 kb, respectively, while each tissue appears to contain a small amount of the other RNA species. These differences most likely reflect organ-specific differences in the extent of clipping at the 5' and/or 3' termini of the spliced, monocistronic clpP mRNA. In the case of rps12, certain transcripts appear to be uniquely present in either stems (0.7 and 2.0 kb) or fruits (2.5, 3.1, 3.3 kb), while the other six or seven transcripts appear to be present in both organs.

Transcript patterns for rp 12

In most photosynthetic land plants, including tobacco (Fig. 2), rp12 is part of a large (ca. 12 genes, ca. 8 kb) operon of mostly ribosomal protein genes [7, 14, 34, 36]. This gene cluster is reduced in size by about 1.5 kb in Epifagus as the result of the deletion of part or all of four of these genes (Fig. 2). As expected, both Epifagus and tobacco show complex transcript patterns for rp12, which also contains a single intron of about 660 bp in both species. As with rps12, certain rpl2 transcripts (of 1.5, 1.9, and 3.1 kb) predominate in tobacco, whereas all rpl2 transcripts are of roughly equal abundance in Epifagus. Compared to transcript levels for the same gene in tobacco, rp12 is notably less highly expressed in Epifagus than are any of the other genes examined (Fig. 1 and data not shown). In spite of these quantita-

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tive differences, several rpl2 transcripts (1.5- 3.1 kb in size) appear to be the same size in Epi- fagus and tobacco.

Transcript patterns for rps2

In striking contrast to clpP, rpsl2 and rpl2, and to the four ribosomal RNA genes, which also show similarly complex transcript patterns in both species (data not shown), the rps2 transcript pattern is much simpler in Epifagus than in tobacco (Fig. 1). In Epifagus, there is a major rps2 transcript of 1.9 kb, a few smaller transcripts, and no larger ones. In tobacco, however, there are many rps2 transcripts, most of which are larger than 2 kb in size. This difference in transcript complexity is an expected consequence of gene loss in Epifagus ptDNA. In photosynthetic plants, rps2, including tobacco (Fig. 2), is upstream of, and cotranscribed with, a cluster of four ATPase genes (atpI, atpH, atpF, and atpA; [ 12, 29]), one of which contains an intron. The first three of these genes are completely absent from Epifagus ptDNA, while only a short remnant of atpA is present (Fig. 2).

Splicing and editing of Epifagus plastid transcripts

To investigate potential splicing and editing of Epifagus plastid transcripts, cDNAs generated from three genes that contain five of the six sur- viving Epifagus plastid introns were analyzed by sizing and sequencing, cDNAs were made by reverse transcription using the 3' primers given in Materials and methods, followed by PCR amplification using the same 3' primers in combination with the indicated 5' primers (primer locations are shown in Fig. 2). Three cDNAs were designed to contain a single intron insertion site (for the single rpl2 intron, and for each of the two clpP introns), while the rps12 cDNA was designed to encompass the positions of both its cis- and trans- spliced introns.

Agarose gel analysis shows that the predomi- nant products from RT-PCR of RNA templates (Fig. 3, lanes 3, 4) are smaller than those from DNA templates (Fig. 3, lanes 1, 2) by amounts corresponding precisely to the sizes of the rel- evant introns (cf. Figs. 2 and 3). For example, the major cDNA product for rpl2 is about 640 bp (Fig. 3, lanes 3, 4), essentially as expected for a spliced transcript (expected size 635 bp; Fig. 2),

Fig. 3. PCR analysis of three intron-containing plastid genes and their transcripts. Primer pairs (see Fig. 2 and Materials and methods for primer locations and sequences) flanking either the single intron in rpl2, both the cis- and trans-spliced introns in rps12, or the first (5') or second (3') intron in clpP were used in PCR amplification of Epifagus total DNA (lanes 1, 2 and 6), total RNA (lanes 3, 4, 7 and 8), or no template (lane 5). Taq polymerase was included in all reactions, whereas a prior reverse transcription step was done for lanes 3, 4 and 8. Lanes 2, 6 and 8 contained RNAase, and lanes 4, 6 and 8 contained DNAase. Sizes (in bp) are shown for some of the bands from the 123 bp size ladder (lanes marked 'M') from GIBCO BRL.

and smaller than the major genomic product of about 1300 bp (Fig. 3, lanes 1, 2; expected size = 1296 bp) by 661 bp, the predicted length of its intron. Similar results hold for the 5' and 3' clpP amplifications. For rps12, the major cDNA product, of about 360 bp (Fig. 3, lanes 3, 4), again corresponds well to the size expected for the spliced gene product (359 bp). The genomic amplifications (Fig. 3, lanes 1, 2), on the other hand, yielded a large array of presumably nonspecific products owing to the trans-spliced organization of its first intron.

In general, the control lanes show that there is no template activity in the reverse transcriptase and PCR reagents (Fig. 3, lanes 5), that production of the DNA-template products of lanes 1 and 2 is DNase-sensitive (lanes 6), and that production of the RNA-template products of lanes 3 and 4 is either reverse transcriptase-dependent (lanes 7) or RNase-sensitive (lanes 8). In addition, lanes 2 show that production of the DNA°template products is RNase-resistant, and lanes 4 that production of the RNA-template products is DNase- resistant. The appearance of an 840 bp band in the negative control lanes 5 and 6 for rpsl2 is, however, unexpected and inexplicable. Overall, these experiments lead us to conclude that significant levels of spliced RNA products are present for all three genes.

The four cDNA PCR products shown in Fig. 3 were completely sequenced directly, without cloning (data not shown). Comparison to the genomic sequences [41] establishes that the five group II introns of these three genes are located and spliced out at exactly the same positions as in the homologous genes of tobacco and other examined land plants [25, 34]. Genomic and eDNA exon sequences were identical for the regions compared in rpl2 and clpP. However, a single difference, indicative of RNA editing, was found for rpsl2. Coding region position 221 is C in the Epifagus plastid genome, but T in the corresponding eDNA. This difference was observed, without any evidence of partial editing (i.e., all of the cDNA PCR products gave homogeneous sequencing ladders), in all five preparations of Epi- fagus RNA and DNA analysed from the same

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plants collected from five locations in Indiana, Michigan and Tennessee. We thus conclude that most or all Epifagus rps12 transcripts, or at least the spliced ones, have undergone a C ~ U editing event. This edit changes codon 74 from TCA (serine) in the gene to UUA (leucine) in the mRNA. Overall, then, only a single edit was found among the total of 1401 bp of cDNA sequence determined for these three genes (this total is the sum of the nine cDNA exon lengths reported in Table 1, minus the overlap in clpP exon 2 between the 5' and 3' clpP cDNAs, and minus the PCR primers).

Discussion

General aspects of Epifagus plastid genome expression

The results presented in this paper firmly support our previous conclusion, based on molecular evolutionary evidence (see Introduction and [41]), that the highly reduced plastid genome of the nonphotosynthetic parasite Epifagus virginia is nonetheless a functional, expressed genome. All 12 Epifagus plastid genes examined are transcribed by the criterion of detection of stable RNAs in northern blot experiments using gene-specific probes. Moreover, eight of the genes display complex transcript patterns reminiscient of those found for the same genes in tobacco and other photosynthetic plants. This indicates that similar processes of transcription initiation and termina- tion from multiple sites, transcript trimming and clipping, and cis- and trans-splicing occur in plastids of photosynthetic and permanently nonphotosynthetic plants. Direct evidence that splicing occurs in Epifagus plastids was obtained by sizing and sequencing cDNAs made by reverse transcriptase-based PCR. These experiments es- tablish that all five examined introns (out of six total in Epifagus ptDNA) are spliced effectively in vivo. Finally, comparison of these cDNA sequences with published genomic sequences [41] reveals that RNA editing, a low-frequency but characteristic process of plastids in photosyn-

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thetic plants [ 18], occurs at similarly low levels in Epifagus plastids.

Overall, then, we conclude that the Epifagus genome is indeed transcribed, and that its transcripts are subject to the entire gamut of post- transcriptional processing events that are well characterized in photosynthetic plastids. The ex- istence of such plastid-characteristic transcript patterns and processing events (e.g., group II intron splicing and RNA editing), together with the identity of the cDNAs to published plastid gene sequences, rules out the formal possibility that the highly reduced plastid genome in Epifagus is actually defunct and that the transcripts detected are instead the products of chloroplast genes transferred to the nucleus. Although we have not formally demonstrated expression of the Epifagus plastid genome at the level of translation, we feel this is a moot point. In our view, the molecular evolutionary evidence - the specific retention of a distinct subset of intact plastid genes (some quite large) against a background of deletion of most genes [41] - taken together with the evidence presented here for transcription and proper post-transcriptional processing of these retained genes, makes it a foregone conclusion that these transcripts are translated into functional plastid polypeptides.

Although seven of the nine genes examined that show complex transcript pattems in tobacco also show complex patterns in Epifagus, in most cases significant qualitative and quantitative differences were observed between the two species using the same gene probe. In other words, the case ofclpP, in which both species show the same-sized pre- dominant transcript, as well as several minor, higher-molecular-weight transcripts of the same or similar sizes, is unusual (Fig. 1). For rps12 and rpl2 (Fig. 1), there are fewer comigrating transcripts in tobacco and Epifagus, and in both cases processing appears to be less efficient in Epifagus, as reflected by a higher proportion of larger to smaller transcripts in the parasite than in tobacco leaf chloroplasts. This last difference may relate more to developmental than evolutionary factors, as it has been shown in maize, for example, that for most plastid genes the ratio of unspliced to

spliced transcripts is much higher in nonphotosynthetic tissues (meristems and roots) than in green leaves [ 1 ]. In this respect, it is important to emphasize that the cDNA analyses shown in Fig. 3 should not be interpreted quantitatively, i.e., they should not be taken to mean that most or all of the transcripts from these three intron- containing genes have been spliced. This is because the much smaller spliced transcripts would be expected to out-compete any unspliced transcripts in both the reverse transcription and PCR-amplification reactions used to generate the cDNAs. Accurate measurement of amounts of spliced and unspliced transcripts requires other approaches, such as the S l-nuclease-protection assay used by Barkan [1].

Some of the qualitative differences seen in transcript patterns between Epifagus and tobacco are, however, an expected consequence of their evolutionary divergence, namely, the extensive gene los s leading to reduction of transcription unit size in Epifagus. At the extreme, far simpler transcript patterns, lacking the abundance of multiple larger transcripts observed in tobacco, were seen for rps2 (Fig. 1) and rpsl4 (data not shown). For both genes, all other genes (four and two, respectively) with which they are cotranscribed in photosynthetic plants are entirely or largely absent from Epifagus ptDNA (Fig. 2; [41]). Operon reduction (from 12 genes in tobacco to eight in Epifagus; see Fig. 2) is also probably responsible for some of the less impressive differences observed in transcript patterns produced by the rpl2 probe (Fig. 1).

RNA editing in Epifagus plastids

A total of 1401 nucleotides of cDNA sequence was determined in this study for the intron- containing genes rps12, rp12 and clpP. Compari- son to the corresponding published [41] plastid gene sequences revealed a single, conventional C ~ U edit. A similarly low incidence of RNA editing has been observed in both photosynthetic and nonphotosynthetic tissues of photosynthetic plants (e.g., [ 19, 23]; reviewed in [ 18]). It is worth

noting that several putative edits were initially observed in comparing cDNA and genomic sequences from Epifagus, but further examination showed that all but one actually reflected popu- lation-level DNA variation. The bona fide rps12 edit is the only difference that persisted when RNAs and DNAs were sequenced from the very same plant. This provides an important caution- ary lesson for all studies purporting to find evidence of RNA editing. The rps12 edit in Epifagus changes codon 74 from TCA (serine) to U U A (leucine). Plastid rps12 genes from 14 other di- verse land plants encode only leucine at this position (J. Allen and J. Palmer, unpublished), implying that the rps12 edit observed in Epifagus evolved relatively recently.

Curiously, the C residue that is edited in Epi- fagus rps12 is embedded in an octanucleotide sequence (ATAATTCA) that is perfectly complementary to part of one of several putative guide RNA consensus sequences identified in plastid and plant mitochondrial genomes by Maier et aL [24]. Furthermore, about 200 bp upstream of this edit site, located within intron 1 of rps12, is a perfect inverted repeat (TGAATTAT) of the edit- octanucleotide sequence. It is tempting to speculate that, prior to splicing, an intramolecular 8-bp stem forms between the edit site and its intronic complement, and that formation of this second- ary structure in the rps12 transcript guides the editing apparatus to this site.

That RNA editing is very rare in the three genes examined in this study, and probably in the Epi- fagus plastid genome as a whole, may be of some functional importance relative to two previous observations regarding the Epifagus plastid genome. First, inferences that virtually all Epifagus plastid gene products have faster rates of sequence evolution than those of photosynthetic plants [27, 41, 42, 44] are supported by the obser- vation that RNA editing is infrequent and thus unlikely to 'correct' extensively modified gene sequences. Second, Epifagus plastid DNA encodes a grossly incomplete set of tRNA genes, but the choice of codons in Epifagus plastid proteins suggests full availability of all tRNAs [27, 42]. Mini- mal RNA editing for these three protein genes

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decreases the likelihood that a full set of tRNAs could somehow be specified from the Epifagus plastid genome by post-transcriptional modifica- tion of its reduced set of tRNA genes and sup- ports our earlier hypothesis of extensive plastid import of cytoplasmic tRNAs [27, 42].

MatK and group H intron splicing in plastids

We have shown that five of the six group II introns in Epifagus p tDNA are spliced properly (we have not examined the sixth, in rp116). These results are significant in light of the curiously freestanding maturase gene, matK, present in Epifa- gus ptDNA. Although freestanding in Epifagus, matK is contained within the group II intron of trnK in all photosynthetic land plants (trnK is absent from Epifagus ptDNA). Mohr et al. [26] showed that MatK (the product of matK), although having lost most of the reverse transcriptase character of group II-encoded proteins, has retained a ca. 100 amino acid domain ('X') characteristic of nearly all group II maturases (i.e., splicing factors) and which they speculate binds to intron RNA. MatK protein was recently detected in potato chloroplasts [6] and has been shown to bind to trnK RNA [22]. Because matK is freestanding in Epifagus, we [41] and others [8, 22, 26] have hypothesized that, unlike other group II maturases, MatK has the novel [20] property of assisting splicing of introns that are distantly related to the one in which it normally resides.

To better understand the evolution and potential functional divergence of MatK in Epifagus, we have analyzed its sequence relatedness to MatK proteins from photosynthetic plants. Epifagus MatK is unusually divergent in two ways. First, as shown in Fig. 4, it is considerably shorter (439 amino acids) than all other MatK proteins (497- 515 amino acids) due to an N-terminal truncation. Second, as is true of virtually all Epifagus plastid gene products [41, 42], MatK is changing faster than normal in Epifagus. Epifagus and tobacco are more closely related to each other than to any of the other species compared in Fig. 4, yet the amino acid identities between MatK of Epi-

730

120

Tob MEEIQRYLQPDR g -QQHNFLYPLIFQEY IYALAHD~OLNR- - - NRg ILL ENPGYNN- KLgFLI'VKRLITRMYQQNHFLISTNDgNKNS FLGCNK8 .... LTgQ~IBEGFAFIV

E~ , .K..L.I..g...KY .FL.-R, .K .... . .TEI.L..LT..L

SUII .O.F.G.. EL.KFR. H. - .................. V...- .... ....D.F.. D-N.g .SI ......... G .... L...A.Y .... K...H..N .... FD ......... V..

pea .K.Y .v..ERS..R. °D-. .... L,R .... G.. YS .HFH.- .... , .F,..V, .D,- ,¥.L ............... LI..A., .P, ,P,W,Y..N .... .DC.I ...... IV,

must . .KF,G..EP.QAR..g-. .... F.R .... V.gY ...... LNR .... P...AD, D-K .T.S ....... L...E..RLI .P .K.L.T.--, .HTNL .... F.Y .... VL..V..

ba~ . .KFEG. S~ZKQK oR..Y-.V,, .L ....... F. • .Y...g ..... . EPV. IVBW..K.F.g.L ..... I ...... FLDN. V.HP.QDRL .DTKIFFTgEP...IL ..... IV.

dee . .KFEG. SEKLKFPR.Y- .V...L ...... VF...Y...G ..... .E.V.II.g..K.F.S.L .... MI ...... FWINLV.HP.QDRL.DT.NP~¥gEF., .IL ..... IV.

~ne . D.FH.CGKE.SPW..C-. .... F.K.DL..Ig...y. DV--~gS.RPM.HLSg .-DQ .... T ..... GQIR .... SZVLFVNCDP. PLADRK o .----F.. EgVL. ALTLVL

Mar I~TILKVLPTYEKQT-NKNFWNKP-YI. IFF,..NF,GI .I'NRFI.KTNYKN--FFFFGLK..--FN..KI .... KKIR, FDFLF.- ......... FgKK.N .... FVNIK ..... EV..

24O Tob EIPFSLRLISBL~gFEGKKIFKSY- -NLR gIHBTFPFLEDNFBHLNYVLDILI PTPVHLEI LVQTLRYWVKDAS BLHLLRFFLHEFWNL~SLIT8 - -KKPGTBFSKKNQRFFFFLTNSTV

Epi .FF.FI .... T---L .... NKIEKLN ..... L.I ......... N.KFI ...... RK..A .......... I. • .TY ...... W. NN-- ,W. ,I. NQ--N.D. • FYPQ. • -KILLL ......

gUll ........ V,, ---L.R.E.V. °H--• ...... I ........ L ..... g ..... H.I .......................... Y. TQSW ..... PTP* .SIgIV.QR. • .L.L .......

pe s .... FRQ. g..---..EAE.L..FK-. ...... I ...... K.TT .... g..R .... I ....... I ........ PFF .... L..TH.C.W..F..T--..gIST. • • S. P.L.L..H.F. •

must ........ G..---....NLK. • .--..Q .... I ...... K...P ..... V ..... I ........... R ........ FF..C.Y. YC.WKNFDgK--..gIL ..... .P. • .L ..... H.

bad ....... EL.C ..... P. EKEIPKFQ ....... I ...... K.L..D.LgH.E .... I ....... L.Q.RIQ.VP ......... NTYS .W..F... --M. SILF. Q. E. K .LVK .......

dGe ....... EQ.C ..... PEEKEIPKFQ ....... I ...... K.L.. H,LAH.E .... I..D. • L. L. Q.RIQ.%'P ......... NYYS .W..F. • .--M.SIFZLK. E,K.L ,R .......

~ne .V...IWSKY.- .... VEGIfl~E. K- -gP ..... I ...... K.P.S.gI.,AR...gI.P .... R. F.R. ZR.. p... p.. SV.y. YR.-- .TENL--QRSIIVVPRV. T.. ,L..W.¥..

Uar I,LDNIFTFE. - .... K. gFI,NKIKgYQ .... I. B.M,HRIYNS..F...T,..FF. P. • .IRIF.RHIQ. IPF..F..TL .Y---KNKC.NIL--NIENFFYL .... -..C..W.F.I

A "160

Tob YEC ESTFVFLRNQg BHLRSTSFGALLERIYFTGKI ERLVEVF- -AKDFQVTLWLFK DPFI~qYVRYQGK~ ILAgKG -TFLLMNKWK FYL%'NFWQCHC~LCFHTGRIHINQLgNHgRDFMGY

Epl CKT, .I ........ Y.P...P.IM ........ F ...... NP.LKV, ° .KAN...I.E.CL..F..RRQ¥ ...... -. 8. FL .... C.VIT...WTF. ,W.FSRS.T.KK,L.N. FEILT.

SuU C.Y .... I..W ...... Q., .T.T.F ......... KH ..... --SN. ,PTAP .......... I ............ -.P..L .... ¥ ......... I"/~WSQg ............ L..L..

pea C.Y.YIL ..... K .... GFK.. SIFF...F .... R.H.GK..--.... gTP.TF .... NI ........ C ..... N-APF ...... H. FIHL. •, FFDVWSQpRM.N..P..E.. FQLL..

mus t C.Y..I.F...K ......... yEVFF...L ..... QHFLK. ,--VNN. PAI.G.L .... L ..... H..T...T.D-.P.M ..... Y. F.. L...YF .VW.QgQK.N ..... KDNFB.L..

bad g. Y. FFLL... K... C. PLAYS. TF .... H. SR .M. HFGIMY--PGFgRK.,. F.M.. LI ........ A ...... -S. F. KK... C.. I .... TYFFFWTOPR ....... A. gCF .....

dGe g .T. FFLL.. ,K., .C..L..g .TF .... I .SR.M.HFGLMY--PAF.RK .I. FVM..L ......... A ...... - .L..KK...C...RL.. TgF. FWTQPQ.. ,L.. ,E.BCF. ,L..

pine C .... ILFgRLKR,. °g. ,L.H.gFPQ ,TH.HR° .KHIIIFg--RRNSLKSI. SL...KI ..... GERP.I. I, °-AH..VK.CRY..LI .R. FTFH.WgEPY .VCSH...KNCSggP..

Mgr .. F.TLLNDIWEKFTKFE °VF .WNFIDKTNSIK.. KHILKKS--K. PIEKKI-VK. ISSI..I..KNNL. ITLNI~NI. ILEN.. DFFLI... KTFRVW ,KSg, .L .QNFTKN. FS .L..

Domain "X"

Tob LgSVRLNPSMVRSQMLE'gFLINNAI~FDTLVPIIPLZGgLAKAN FcTVLGHPIgKPVWSDLgD"DIIDRFGRIcRNLFHTYSGSSKKKTL'RIKYILRL"c"TLARKHKgTVRT4:o- Ep] H..LNI...F.L* .I, .TP.Ig..T...VNI ..... LS.--SD.EK.FN ..... T. NLFRV .... YN..G.LEC .Y,, .Y..L ...... NS..L ..... L, • .V .............. AF

SUIt ......... V..N ....... I.D ....... II. • .L. • .R ..... K..N.V.D ..... S.AES ......... V ..... IS. °H. ,A .... S ......................... A.--

pea F.N .... R.V ...... Q.T...EIV...L.II ....... R ..... K..N...E ....... A.8..F ...... L ...... g...N ...... S ............ IK...C ....... A.--

must F..L...SLV ............ D,VRR .L. SNIQ. gsI ...... DK..N ........ AT.M. S ..... LN..V ..... IS ...... A,..N .......... C.VK ........... A.--

bad .... pKg. LL. °N ......... DTRM ..... I..ATL., .Y.g..Q...GB ....... I .T .... W..L ............. H ...... R .... L ....................... --

dce F...PI.SLL..N ......... DTQM ..... K..~rT ......... Q...Gg ....... I.T .... W..L ............. H ........... L ..................... A.--

~R@ FLR..M.. IL..TK.. DEL. IADLITDEI. PI.. ,V. I.. L, .TEK. • DIB.R .... LS.Tg.T.D..L... DQ.W .......... FI~DG ........ g .... K...C ..... I.~r~--

Mar MFRIESQIILIQI. IINLLRNV.LIK. E. CgZI.V .... RL...EK..D...R.LC .Lg .TT., .Ng.FE.. DQ.IKHI.S .... c'rN..G, .QLQ..F,F...K...C ..... I..V--

| 540 Tob - L K R ~ g E - L L ~ - - FLTS W.F~VLBL'I'FPR.AB - g g L W G V ' f R g R T W T L D T F C ~ - YQ 509 100% 60% EpI QK..LCL.-...O--. FI... DI. F.K,QKVT-...O...I.OF...M.Tg..T. • 439 60% 100%

Su|l -...L, .G-., • .--.. ,ED..A.. ,I ..... -.T, .RL..G ....... I ..... V.-HE 506 77% 53%

peg -. ...... E, ,Q.--. F.E, .EII. ,I.., D, -.T.QRLH.N ...... 495 67% 49%

must -.. ,L, .G-....--...G.D ..... Z. •. SD-YASKRL..V.V ..... LTL...V.-HE 502 65% 49%

bad -MQ. L..A-F...-- .F.E .... F, .M.TKTTLF. FB. BHTEPY ..... IG .... V.pLN 511 60% 46%

d©e -MQWL.. V-F... - -. F. E .... F.. M. AKTTYF. FR. ffH fie ....... LR .... V. PLN $I I 60% 44%

pine -R. EL, p.-. FKK--gFgK.REFD..P ,SSKA---AARgQ.E... HS.. PQ,. P... SW.KIQDLKIGNLFDQ 51S 44% 38% Mar -w.xY..N-.. TBSI. FNK-TKL "r . .N. gNK ...... NP. KKNF... N.'rQv. T.. HgL.KgKLLKE 497 34% 33%

Fig. 4. Unusual divergence of MatK in Epifagus. An alignment of MarK amino acid sequences from tobacco (tob [33]), Epifagus virginiana (Epi [41]), Sullivantia sullivantii (Sull [ 13]), pea [2], mustard (must [28]), barley (barl [3, 31 ]; GenBank accession number X64129), rice [11], Pinus contorta (pine [21]), and Marchantia polymorpha (Mar [38]) was first made using CLUSTAL [9] and then improved by eye. Dots represent identical amino acids; dashes represent gaps. The bracketed region is the ca. 100 amino acid long domain X, a putative RNA-binding domain common to almost all group II intron maturases [26]. The length of each protein is given in the first column following the alignment, followed by the percent amino acid identity (gaps excluded) to MatK from tobacco (second column) and Epifagus (third column). This alignment contains several adjustments compared to published inferences of the MatK start site. Completion [41 ] of the partial marK sequence of Morden et al. [27] reveals that the 439 amino acid long Epifagus MatK probably starts at a position 200 bp downstream from the tobacco start site; translation of the Epifagus sequence from the tobacco start site yields a stop codon 60 codons downstream of this ATG as a consequence of a short frameshift mutation in Epifagus. Although Mohr et al. [26] extended the inferred length of Epifagus MatK at 505 amino acids by postulat- ing an error in our published [41] matK sequence that would eliminate this frameshift, reinspection of our sequencing films con- firms the frameshift, and we therefore stand by our original inference that Epifagus MarK is 439 amino acids in length. In accor- dance with the proposal ofTsudzuki et aL [37], the reported [38] 370 amino acid MatK (see arrow) of Marchantia has been extended to 497 amino acids via introduction of an extra T in a run of 12 T's at positions 26, 745-26, 756. The published reading frames of the sequences from mustard [28] and rice [11 ] begin 22 and 31 codons, respectively, before the start sites proposed in Fig. 3. However, the nucleotide sequence of this region is mustard contains five frameshift insertions of 1, 2 or 5 bp relative to tobacco,

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fagus and other angiosperms (e.g., Epifagus-pea, 49 ~o) are much lower than between tobacco and the same plants (i.e., tobacco-pea, 67 ~o; see val- ues at end of Fig. 4). Despite its rapid sequence divergence and unusual N-terminal truncation, Epifagus MatK is nonetheless highly colinear with other plastid MatK proteins (Fig. 4).

If MatK is indeed a maturase in Epifagus, then which of the six group II introns still present in its genome might it act on? Two fines of evidence suggest that the single group II intron in rpl2 is a good candidate for being a site of MatK action in Epifagus. First, Hess et al. [8] showed that the rpl2 intron is the only one of seven group II introns examined whose splicing is blocked in four different nuclear mutants of barley, each of which is severely deficient in its level of plastid ribo- somes. They concluded that a functioning plastid translational apparatus is necessary, most likely to produce MatK, in order for splicing of the rpl2 intron to occur. Second, although still distantly related overall, the rpl2 intron is nonetheless the most closely related plastid intron in sequence to the intron in trnK (the host gene for matK in photosynthetic plants), based both on formal phylogenetic analysis (data not shown) and on the classification scheme of Michel et al. [25]. A second candidate for MatK-assisted splicing in Epifagus is the cis-spliced intron 2 of rpsl2, whose splicing has also been shown to be blocked in plastid ribosome-deficient mutants of barley (T. BOrner, W.R. Hess and T. Htlebschmann, pers. comm.). Of the other four group II introns present in Epifagus, two (the trans-spliced intron 1 ofrpsl2 and the single intron in rpll6) are spliced in the barley mutants (and are therefore probably not dependent on MatK for their splicing; [8], T. BOrner, W.R. Hess and T. Htiebschmann, pers. comm.), while the two clpP introns could not be analyzed because they are absent from the barley clpP gene (S. Downie and J. Palmer, unpublished). In summary, we suggest that there are

two good candidates for MatK action in Epifa- gus: the single intron in rpl2 and the second intron in rpsl2.

Acknowledgements

We are grateful to Thomas BOrner, Wolfgang Hess, Thomas H~lebschmann, Karsten Liere, and Gerhard Link for communicating their unpublished results; Thomas BOrner, L. Michelle Bowe, and B. Swalla for their comments on the manu- script; and John Mullet for providing a clone of barley marK for our reanalysis of its sequence (see GenBank accession number X64129). This work was supported by funds from the NIH (GM-35087) to J.D.P., the Alfred P. Sloan Foun- dation (90-3-5) to K.H.W. and the NSF (DEB- 9120258) to C.W.D.

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